Detergent-based decellularized peripheral nerve allografts: An in vivo preclinical study in the rat sciatic nerve injury model.

Nerve autograft is the gold standard technique to repair critical nerve defects, but efficient alternatives are needed. The present study evaluated the suitability of our novel Roosens-based (RSN) decellularized peripheral nerve allografts (DPNAs) in the repair of 10-mm sciatic nerve defect in rats at the functional and histological levels after 12 weeks. These DPNAs were compared with the autograft technique (AUTO) and Sondell (SD) or Hudson (HD) based DPNAs. Clinical and functional assessments demonstrated a partial regeneration in all operated animals. RSN-based DPNAs results were comparable with SD and HD groups and closely comparable with the AUTO group without significant differences (p > .05). Overall hematological studies confirmed the biocompatibility of grafted DPNAs. In addition, biochemistry revealed some signs of muscle affection in all operated animals. These results were confirmed by the loss of weight and volume of the muscle and by muscle histology, especially in DPNAs. Histology of repaired nerves confirmed an active nerve tissue regeneration and partial myelination along with the implanted grafts, being the results obtained with HD and RSN-based DPNAs comparable with the AUTO group. Finally, this in vivo study suggests that our novel RSN-based DPNAs supported a comparable tissue regeneration, along the 10-mm nerve gap, after 12-week follow-up to HD DPNAs, and both were superior to SD group and closely comparable with autograft technique. However, further improvements are needed to overcome the efficacy of the nerve autograft technique.

Impact of modified gelatin on valvular microtissues.

A significant challenge in the field of tissue engineering is the biofabrication of three-dimensional (3D) functional tissues with direct applications in organ-on-a-chip systems and future organ engineering. Multicellular valvular microtissues can be used as building blocks for the formation of larger scale valvular macrotissues. Yet, for the controlled biofabrication of 3D macrotissues with predefined complex shapes, directed assembly of microtissues through bioprinting is needed. This study aimed to investigate if modified gelatin is an instructive material for valvular microtissues. Valvular microtissues were encapsulated in modified gelatin hydrogels and cross-linked in the presence of a photoinitiator (Irgacure 2959 or VA-086). Hydrogel properties were determined, and valvular interstitial cell functions like phenotype, proliferation, migration, mRNA expression of extracellular matrix (ECM) molecules, ECM deposition, and tissue fusion were characterized by histochemical stainings and RT-qPCR. Encapsulated microtissues remained viable, produced heart valve-related ECM components, and remained in a quiescent state. However, encapsulation induced some changes in ECM formation and gene expression. Encapsulated microtissues showed lower remodeling capacity and increased expression levels of Col I/V, elastin, hyaluronan, biglycan, decorin, and Sox9 compared with nonencapsulated microtissues. Furthermore, this study demonstrated that proliferation, migration, and tissue fusion was more pronounced in softer gels. In general, we evidenced that modified gelatin is an instructive material for physiologically relevant valvular microtissues and provided a proof of concept for the formation of larger valvular tissue by assembling microtissues at random in soft gels.

Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves.

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.

Complete Static Repopulation of Decellularized Porcine Tissues for Heart Valve Engineering: An in vitro Study.

To date, a completely in vitro repopulated tissue-engineered heart valve has not been developed. This study focused on sequentially seeding 2 cell populations onto porcine decellularized heart valve leaflets (HVL) and pericardia (PER) to obtain fully repopulated tissues. For repopulation of the interstitium, porcine valvular interstitial cells (VIC) and bone marrow-derived mesenchymal stem cells (BM-MSC) or adipose tissue-derived stem cells (ADSC) were used. In parallel, the culture medium was supplemented with ascorbic acid 2-phosphate (AA) and its effect on recolonization was investigated. Subsequently and in order to obtain an endothelial surface layer similar to those in native HVL, valvular endothelial cells (VEC) were seeded onto the scaffolds. It was shown that VIC efficiently recolonized HVL and partially also PER. On the other hand, stem cells only demonstrated limited or no subsurface cell infiltration of HVL and PER. Interestingly, the addition of AA increased the migratory capacity of both stem cell populations. However, this was more pronounced for BM-MSC, and recolonization of HVL appeared to be more efficient than that of PER tissue. VEC were demonstrated to generate a new endothelial layer on HVL and PER. However, scanning microscopy revealed that these endothelial cells were not allowed to fully spread onto PER. This study provided a proof of concept for the future generation of a bioactive tissue-engineered heart valve by showing that bioactive HVL could be generated in vitro within 14 days via complete repopulation of the interstitium with BM-MSC or VIC and subsequent generation of an entirely new endothelium.

Scaffold-free high throughput generation of quiescent valvular microtissues.

AIMS: The major challenge of working with valvular interstitial cells in vitro is the preservation or recovery of their native quiescent state. In this study, a biomimetic approach is used which aims to engineer small volume, high quality valve microtissues, having a potential in regenerative medicine and as a relevant 3D in vitro model to provide insights into valve (patho)biology. METHODS AND RESULTS: To form micro-aggregates, porcine valvular interstitial cells were seeded in agarose micro-wells and cultured in medium supplemented with 250µM Ascorbic Acid 2-phosphate for 22days. Histology showed viable aggregates with normal nuclei and without any signs of calcification. Aggregates stained strongly for GAG and collagen I and reticular fibers were present. ECM formation was quantified and showed a significant increase of GAG, elastin and Col I during aggregate culture. Cultivation of VIC in aggregates also promoted mRNA expression of Col I/III/V, elastin, hyaluronan, biglycan, decorin, versican MMP-1/2/3/9 and TIMP-2 compared to monolayer cultured VIC. Phenotype analysis of aggregates showed a significant decrease in α-SMA expression, and an increase in FSP-1 expression at any time point. Furthermore, VIC aggregates did not show a significant difference in OCN, Egr-1, Sox-9 or Runx2 expression. CONCLUSION: In this study high quality valvular interstitial cell aggregates were generated that are able to produce their own ECM, resembling the native valve composition. The applied and completely cell driven 3D approach overcomes the problems of VIC activation in 2D, by downregulating α-SMA expression and stimulating a homeostatic quiescent VIC state.

Impact of Detergent-Based Decellularization Methods on Porcine Tissues for Heart Valve Engineering.

To date an optimal decellularization protocol of heart valve leaflets (HVL) and pericardia (PER) with an adequate preservation of the extracellular matrix (ECM) is still lacking. This study compares a 4 day Triton X-100-based protocol with faster SDC-based protocols for the decellularization of cardiac tissues. Decellularized and non-treated HVL and PER were processed for histological, biochemical and mechanical analysis to determine the effect of these agents on the structure, ECM components, and biomechanical properties. Tissues treated with SDC-based protocols still showed nuclear material, whereas tissues treated with Triton X-100 1% + ENZ ± TRYP were completely cell free. For both decellularized tissues, an almost complete washout of glycosaminoglycans, a reduction of soluble collagen and an alteration of the surface ultrastructure was observed. Interestingly, only the elastic fibers of pericardial tissue were affected and this tissue had a decreased maximum load. This study showed that both detergents had a similar impact on the ECM. However, Triton X-100 1% +DNase/RNase (ENZ) ± Trypsin (TRYP) is the only protocol that generated completely cell free bioscaffolds. Also, our study clearly demonstrated that the decellularization agents have more impact on pericardial tissues than on heart valve leaflets. Thus, for the purpose of tissue engineering of heart valves, it is advisable to use valvular rather than pericardial matrices.

Non-Cytotoxic Crosslinkers for Heart Valve Tissue Engineering.

BACKGROUND AND AIM OF THE STUDY: Currently, no effective crosslinking reagents are available to treat xenogenic decellularized heart valve matrices. The study aim was to evaluate the crosslinking effect of quercetin, catechin, caffeic acid and tannic acid on porcine aortic valve matrices. METHODS: Cytotoxicity of the different crosslinkers was evaluated. The mechanical properties of crosslinked porcine matrices and control matrices (non-fixed) were examined by tensile strength testing, as was the cytocompatibility of the fixed matrices. Crosslinked and control matrices were implanted subcutaneously in Wistar rats (n = 9) and, after two weeks, their calcium contents were determined using inductively coupled plasma-mass spectrometry. The antibody reaction against porcine tissue in rat serum was also determined. RESULTS: Cytotoxicity studies showed that crosslinkers, even at high concentrations, did not inhibit cell viability. All crosslinkers except tannic acid improved the mechanical strength of acellular porcine matrices. Moreover, the tensile strength of quercetin-fixed matrices was comparable with that of glutaraldehyde (GTA)-fixed leaflets. Light microscopic evaluation showed that crosslinked matrices caused only a mild lymphocytic inflammatory reaction. Furthermore, quercetin-fixed leaflets exhibited a well-preserved matrix without infiltration of CD3+ cells. After two weeks, calcium levels were 206.33 µg/mg for controls (non-fixed), and 151.33 µg/mg, 181 µg/mg and 163.66 µg/mg for quercetin-, catechin-, and caffeic acid-fixed matrices, respectively. At two weeks after implantation the quercetin-crosslinked matrices also elicited the lowest levels of IgG antibodies. CONCLUSION: The study results identified quercetin as the most suitable crosslinker for heart valve tissue engineering, and a possible alternative to GTA. Further studies are essential to determine whether quercetin crosslinking will allow autologous cell repopulation in order to create a viable heart valve.